Effect of triphenylphosphonium moiety on spatial structure and biointeractions of stereochemical variants of YRFK motif (original) (raw)

2018, European Biophysics Journal

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Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain

Protein science : a publication of the Protein Society, 2017

The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with non-phosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyses along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were carried out. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different confor...

Depalmitoylation of Ykt6 Prevents its Entry into the Multivesicular Body Pathway

Traffic, 2008

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo. The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and -in contrast to the wild-type proteindoes not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.

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